The invention relates to the treatment of hematologic disorders, e.g., disorders characterized by unwanted cells of hematopoietic origin, e.g., hematologic cancers.
Bone marrow transplantation (BMT) has yet to realize its full potential for the treatment of hematologic malignancies. A major obstacle to further advancement is graft-versus-host disease (GVHD), which has been prevented by removing T cells from the donor marrow. Unfortunately, T cell depletion has been associated with increased rates of engraftment failure and leukemic relapse. Despite improvements in pharmacologic GVHD prophylaxis, severe acute and chronic GVHD are still major complications of HLA-matched sibling bone marrow transplantation. Immunosuppressive drugs used for GVHD prophylaxis may also increase the relapse rate for certain types of leukemia. The patients receiving allogeneic BMT are, nevertheless, a fortunate select group: most patients do not have an HLA-matched sibling or a phenotypically matched unrelated donor, and therefore do not have the option of BMT. Attempts to perform BMT between strongly HLA-mismatched donor-recipient pairs have been associated with a prohibitively high incidence of severe GVHD and of failure of engraftment. Furthermore, a large fraction of leukemias and lymphomas afflict older patients who are more prone to the development of GVHD than are younger persons, and who therefore are not generally considered candidates for BMT, despite the lack of other curative options.
The inventors have discovered that hematologic disorders, e.g., both neoplastic (hematologic cancers) and non-neoplastic conditions, can be treated by the induction of mixed chimerism in the absence of whole body irradiation (total myeloablation protocols) or other myeloablative treatment. Methods of the invention reduce GVHD, especially GVHD associated with mismatched allogeneic or xenogeneic donor tissue, yet provide significant graft-versus-leukemia (GVL) effect and the like.
Certain embodiments of the subject methods also feature preparative regimens which minimize or eliminate the need for myeloablative treatment, e.g., hematopoietic space-creating irradiation, especially, preparative whole body irradiation.
One aspect of the present invention provides a method for treating a subject having a hematologic disorder comprising: (i) administering a myeloreductive nonmyeloablative treatment to the subject in sufficient amount such that mixed hematopoietic chimerism can be induced in the subject, and (ii) introducing into the subject, allogeneic donor hematopoietic stem cells (donor stem cells) to form chimeric bone marrow in the subject.
In certain embodiments, the myeloreductive treatment includes treating the subject with an immunosuppressant regimen, prior to introduction of the donor stem cells, in an amount sufficient to prevent rejection of the donor stem cells.
Likewise, the method can include a further step of treating the subject with an immunosuppressant regimen, after introduction of the donor stem cells, in an amount sufficient to prevent a graft-versus-host response mediated by the donor stem cells.
Such immunosuppressant regimens can include, independently for pre- and post-transplantation is both are carried out, a treatment of the subject which inactivates and/or depletes host T-lymphocytes and/or natural killer (NK) cells in the subject. For example, the immunosuppressant regimen includes treatment with T cell-depleting anti-CD4 and/or CD8 antibodies, such as anti-thymocyte globulin (ATG), OKT3 (Orthoclone OKT3 monoclonal antibody, Ortho Pharmaceutical Corp), LO-CD2a (U.S. Pat. No. 5,730,979), or Minnesota anti-lymphoblast globulin (MALG). Preferably, the immunosuppressant regimen, both before and after transplantation, includes administration of ATG.
Moreover, the immunosuppressant regimen can include treatment with thymic irradiation. Preferably, the pre-transplantation immunosuppressant conditioning includes administration of ATG and thymic irradiation.
In other embodiments, the immunosuppressant regimen includes treatment with one or more of a macrolide immunosuppressant, azathioprine, steroids (e.g., prednisone, methyl prednisolone), sub-lethal nonmyeloablative irradiation of lymphocyte-containing tissue, or costimulatory blocking agents (e.g., anti-CD40 ligands, CTLA4Ig fusion proteins, see, e.g., Lenschow et al., (1992) Science 257:789; and Turka et al., (1992) PNAS 89:11102).
In certain embodiments, the myeloreductive treatment includes treating the subject, prior to introduction of the donor stem cells, with an cytoreductive agent selected from one or more of alkylating agents (e.g., nitrogen mustards [such as mechloretamine], cyclophosphamide, melphalan and chlorambucil), alkyl sulphonates (e.g., busulphan), nitrosoureas (e.g., carmustine, lomustine, semustine and streptozocine), triazenes (e.g., dacarbazine), antimetabolites (e.g., folic acid analogs such as methotrexate), pyrimidine analogs (e.g. fluorouracil and cytarabine), purine analogs (e.g., fludarabine, idarubicin, cytosine arabinoside, mercaptopurine and thioguanine), vinca alkaloids (e.g., vinblastine, vincristine and vendesine), epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitomycin), dibromomannitol, deoxyspergualine, dimethyl myleran and thiotepa.
Preferably, the myeloreductive treatment includes treating the subject with cyclophosphamide.
Preferably, the pre-transplantation conditioning includes administration of ATG and cyclophosphamide, and thymic irradiation. Preferably the cyclophosphamide, or other cytoreductive agents, are substantially cleared from the patient so as not inhibit proliferation of the transplanted stem cells.
An important use of the subject method is for allogeneic transplantation of donor stem cells which are mismatched, with respect to the subject, at one or more HLA class II antigens.
Another important use of the subject method is for allogeneic transplantation of donor stem cells which are mismatched, with respect to the subject, at two or more HLA antigens (either HLA class I or II or both).
In preferred embodiments, the donor stem cells are provided as allogeneic bone marrow, mobilized peripheral blood cells, or cord blood cells.
The donor stem cells, in some instances, can be expanded ex vivo for transplantation.
In preferred embodiments, the donor stem cells are from the same species as the subject. However, the present application also specifically contemplates that the donor stem cells are xenogeneic stem cells from a different species than the subject. In xenogeneic methods, the subject is a mammal, preferably a primate and more preferably a human. The donor mammal can be, by way of example, a swine, e.g., a miniature swine, or a nonhuman primate. In xenogeneic methods the donor of stem cells and the donor of leukocytes need not be the same individual but can be from different individuals which are MHC matched or highly inbred, e.g., inbred miniature swine which are MHC matched.
In preferred embodiments, the subject is a human, and even more preferably, the subject is a human and donor stem cells are from another human.
The methods of the present invention can be used to treat a wide range of hematologic disorders, including neoplastic proliferation of hematopoetic cells, such as lymphoblastic leukemia, myelogenous leukemia, Hodgkin""s lymphoma, Non-Hodgkin""s lymphoma and myelodysplastic syndrome. As described herein, the subject method can be used to treat hematologic disorders which are refractory to chemotherapy, such a chemorefactory Non-Hodgkin""s lymphoma.
In other embodiments, the subject method can be used to treat hematologic disorders which are non-malignant, such as erythrocyte abnormalities or immune system disorders. For example, the instant method can be used to treat hemoglobinopathies, e.g., sickle cell anemia, aplastic anemia or thalassemia. The subject method also can be used as part of a treatment regimen for autoimmune disorders as well as immunodeficiencies.
In several embodiments, particularly where little, and preferably no GVHD is detected post-transplantation (e.g., at least 14 days, and more preferably at least 25, 30 or even 35 days), the subject method includes the further step of administering allogeneic donor leukocytes to the subject after introduction of the donor stem cells. The administration of donor leukocytes should be delayed sufficiently from the time of any hematopoietic space creating treatment such that the level of pro-inflammatory cytokines induced by the space creating treatment has subsided sufficiently to reduce or substantially eliminate GVHD from the donor leukocytes.
The subject method can also include the management of GVHD responses post-transplantation by administration of immunosuppressants, or by use of engineered stem cells which give rise to small molecule ablatable T cells or other hematopoietic cells. See, for example, U.S. Pat. No. 5,834,266.
In another aspect, the invention features a method of treating a non-neoplastic disorder or a hemoglobinopathy, e.g., sickle cell anemia, aplastic anemia, thalassemia Thus, in one preferred embodiment, the subject method comprises: (i) identifying a patient having a neoplastic hematopoetic disorder, (ii) administering a myeloreductive treatment to the subject in sufficient amount such that mixed hematopoietic chimerism can be induced in the subject, and (iii) introducing into the subject, allogeneic donor hematopoietic stem cells (donor stem cells) to form stable mixed chimeric bone marrow in the subject.
In another preferred embodiment, the subject method comprises: (i) identifying a patient having a neoplastic hematopoetic disorder, (ii) administering a myeloreductive treatment to the subject in sufficient amount such that mixed hematopoietic chimerism can be induced in the subject, and (iii) introducing into the subject, allogeneic donor hematopoietic stem cells (donor stem cells) to form mixed chimeric bone marrow in the subject, wherein the donor stem cells are mismatched, with respect to the patient, at one or more class II HLA antigens.
In still another preferred embodiment, the subject method comprises: (i) identifying a patient having a neoplastic hematopoetic disorder, (ii) administering a myeloreductive treatment to the subject in sufficient amount such that mixed hematopoietic chimerism can be induced in the subject, and (iii) introducing into the subject, allogeneic donor hematopoietic stem cells (donor stem cells) to form mixed chimeric bone marrow in the subject, wherein the donor stem cells are mismatched, with respect to the patient, at two or more HLA antigens, e.g., class I and/or class II.
In yet another preferred embodiment, the subject method comprises: (i) identifying a patient having a neoplastic hematopoetic disorder, (ii) administering a myeloreductive treatment to the subject in sufficient amount such that mixed hematopoietic chimerism can be induced in the subject, (iii) introducing into the subject, allogeneic donor hematopoietic stem cells (donor stem cells) to form mixed chimeric bone marrow in the subject, and (iv) administering a post-transplantation immunosuppression regimen for suppressing or depleting T-cells in the transplanted donor stem cells.
In yet another preferred embodiment, the subject method comprises: (i) identifying a patient having a neoplastic hematopoetic disorder, (ii) administering a pre-transplantation conditioning to the subject in sufficient amount such that mixed hematopoietic chimerism can be induced in the subject, which pre-transplantation conditioning includes treating the cells with cyclophosphamide, ATG and thymic irradiation in an amount sufficient to reduce rejection of transplanted donor stem cells; and (iii) introducing into the subject, allogeneic donor hematopoietic stem cells (donor stem cells) to form mixed chimeric bone marrow in the subject, and (iv) administering ATG to the subject post-transplant for suppressing or depleting T-cells in the transplanted donor stem cells.
Another aspect of the present invention relates to the use of donor allogeneic stem cells in the manufacture of a medicament for the treatment of a hematologic disorder, wherein the medicament administered to a patient conditioned with myeloreductive non-myeloablative treatment, and in an amount sufficient to form chimeric bone marrow in the subject.
Still another aspect of the present invention provides a kit for allogeneic hematopoietic stem cell transplantation. The kit includes cyclophosphamide in an amount sufficient to reduce rejection of transplanted donor stem cells when administered to a patient pre-transplantation, and ATG in an amount sufficient to reduce rejection of transplanted donor stem cells when administered to a patient pre-transplantation and suppress T-cells in transplanted donor stem cells. The kit may also include a labeled antibody for detecting leukocytes as part of a step of determining chimerism of a treated animal. The kit may also include HLA-mismatched donor stem cells, e.g., allogeneic BMT, mobilized peripheral blood cells, cord blood cells, or hematopoietic cells derived from cultured stem/progenitor cells.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).